Establishment and Application of the LAMP Detection Method for Decapod Iridescent Virus 1 (DIV1)

Abstract:

The loop-mediated isothermal amplification (LAMP) detection method of Decapod iridescent virus 1 (DIV1) was established and optimized based on primers designed from the DIV1 capsid protein gene in present study. Analytic sensitivity of the newly established method was assessed using the pMD18-DIV1 plasmid standard as a template, and the detection specificity was also determined. The results showed that the optimal reaction temperature for the DIV1-LAMP method was 64.4℃, and the optimized 25 μl reaction system contained 2.5 μl 10×isothermal amplification buffer, 4.0 mmol/L Mg2+, 1.2 mmol/L dNTPs; 6.4 U Bst 2.0 WarmStart® DNA polymerase and 0.8 μmol/L EvaGreen®. The detection limit of the new method was 3.54×102 copies/reaction; it did not cause a cross-reaction with major shrimp pathogens such as Enterocytozoon hepatopenaei (EHP), Vibrio causing acute hepatopancreatic necrosis disease (VpAHPND), covert mortality nodavirus (CMNV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syndrome virus (WSSV), Taura syndrome virus (TSV), or yellow head virus (YHV). The newly developed method showed good repeatability and stability. The on-site, rapid, highly sensitive detection method of DIV1 was established by replacing EvaGreen® with GeneFinder® which was preset in the cap of the reaction tube. The DIV1-LAMP real-time fluorescence and on-site LAMP detection method established in this study is sensitive, specific, and rapid. This novel method will provide new technical options for the qualitative, quantitative, and rapid on-site detection of the emerging DIV1, which will benefit the shrimp farming industry by facilitating the monitoring, early warning, and prevention of DIV1.